Week 8 - enzyme interactions

Question 1

Define what an enzyme is

Answer 1

• Specific protein catalysts that increase the rate of chemical reactions without being consumed by the reaction

Question 2

How may enzymes be targets for therapeutic drugs?

Answer 2

• drugs could act as an alternative substrate for the enzyme competing with the specific substrate for binding to the active site and in general inhibiting its activity
• Alternatively, a drug could act by inhibiting the enzymes activity through binding to other sites on the enzyme in allosteric modulation

Question 3

Besides the inhibition and modulation of an enzyme, how else may drugs interact with an enzyme?

Answer 3

• Some drugs must undergo modification to convert thm from the inactive (prodrug) form to an active form

Question 4

What is the key physical characteristic of drugs?

What impact does this have on the body?

Answer 4

• Drugs, by design, are lipophillic which ensures that they can be readily absorbed from the GI tract and taken up into the circulation
• However, the lipophillicity means that they would tend to be continually reabsorbed in the kidney tubules where the tissue is more oil-rich than the urine
• This would result in the accumulation of toxins in cell membranes causing damage or prolonged pharmacological activity
• How this is avoided is discussed in card #5

Question 5

How are the issues arising from a drugs lipophillic nature avoided?

Answer 5

• Lipophillic substances undergo biotransformation to make them mor ehighly water soluble
• Some drugs, at physiological pH 7.4, exist in their fully ionised form and can be secreted without any modifications
• Most drugs undergo sequential metabolic changes categorised as phase I and phase II reactions

Question 6

Briefly describe which group of enzymes is responsible for phase I metabolism of drugs

How must this be considered in drug discovery?

Answer 6

• CYP450 group are the most abundant enzymes in the liver and are responsible for the metabolism (phase I) of 70% of drugs in the body
• Dominant CYP in the liver is CYP3A4, which is 70% of all the CYP450 enzymes in the liver, which has a broad substrate specificity meaning it metabolises a vast range of molecules
• Screening of the CYP450 enzymes is important in early drug discovery

Question 7

Define “drug metabolism” or “xenobiotic metabolism”

Answer 7

• Drug / xenobiotic metabolism is the chemical modification of substances, including drugs or xenobiotics such as toxic chemicals, through the action of a specific family of enzymes
• They convert lipophillic/hydrophobic substances that can acumulate in lipid membranes into more hydrophillic metabolites which can be excreted from the body

Question 8

Define “xenobiotic”

How are xenobiotics metabolised?

Why is metabolism required?

Answer 8

• A xenobiotic is a chemical compound, such as drug, pesticide, carcinogen, that is foreign to a living organism and enters from the external environment
• They are metabolised by pathways of biotransformation that can be found in most organisms, including bacteria which suggests that these pathways have ancient origin
• These reactions are required to detoxify poisonous compounds and remove them from the body before they can cause harm

Question 9

What does the rate of drug metabolism have on a drug?

Answer 9

• Rate at which the conversion from a lipophillic/hydrophobic substance to a more hydrophillic substance impacts the duration and intensity of a drug’s pharmacological activity

Question 10

How many phases does drug metabolism take place in?

Describe these phases

Answer 10

• Drug metabolism takes place in three phases
• Phase I involves enzymes, such as cytochrome P450 oxidases, which make xenobiotics more hydrophillic and reactive by introducing polar groups into their structures
• Once modified, nucleophile metabolites are conjugated to polar compounds through glucuronidation, acetylation or sulphonation and electrophile metabolites are modified by glutathione conjugation in phase II reactions which are catalysed by transferase enzymes such as glutathione S-transferases
• In phase III reactions, efflux transporters recognise the conjugated metabolites and use the hydrolysis of ATP to pump them out of the cell against the concentration gradient

Question 11

Outline phase I metabolism of xenobiotics

Answer 11

• Phase I involves enzymes, such as cytochrome P450 oxidases, which make xenobiotics more hydrophillic and reactive by introducing polar groups into their structures

Question 12

Outline phase II metabolism of xenobiotics

Give an example of the enzyme involved

Answer 12

• Once modified, nucleophile metabolites are conjugated to polar compounds through glucuronidation, acetylation or sulphonation and electrophile metabolites are modified by glutathione conjugation in phase II reactions
• Catalysed by transferase enzymes such as glutathione S-transferases

Question 13

Outline phase III metabolism of xenobiotics

Answer 13

• In phase III reactions, efflux transporters recognise the conjugated metabolites and use the hydrolysis of ATP to pump them out of the cell against the concentration gradient

Question 14

Give an overview of the cytochrome P450 enzymes, what they catalysed and what they are responsible for

Answer 14

• Cytochrome P450 (CYP450) superfamily are a group of enzymes that catalyse the oxidation of lipid soluble xenobiotics
• Role if CYP450 enzymes is to catalyse the metabolism of endogenous substances such as lipids and steroidal hormones, as well as exogenous xenobiotics including drugs and toxic chemicals

Question 15

Outline a typical reaction catalysed by CYP450 enzymes

Answer 15

• Typical reaction catalysed by CYP450 enzymes is a monooxygenase reaction
• This involves the binding of substrate and molecular oxygen to CYP and the tranfer of one oxygen atom into a substrate (RH) while the second oxygen atom is reduced to water
• RH + O₂ + NADPH + H⁺ → ROH + H₂O + NADP⁺

Question 16

Outline the structure and location of the CYP450 enzymes

Answer 16

• CYP450’s are membrane bound haemoproteins that are located in the smooth endoplasmic reticulum
• In the liver they are often closely associated with enzymes wich catalyse phase II conjugation reactions, e.g. UDP-glucuronyl tranferase (UDPGT)

Question 17

How many CYP enzymes are there in humans?

How are they classified?

Answer 17

• There are a large number of CYPs in nature and 57 CYPs in humans, of which 15/57 are responsible for metabolising drugs and other xenobiotics
• Classified on their amino acid homology and can be divided into families
  
  • 18 families have been identified in humans (CYP1-18)
  • There are subfamilies based on sequence homology and are termed CYP1A, CYP2A/B/C/etc.

Question 18

How is substrate specificity of CYP isoforms determined?

Answer 18

• Specificity of the CYP isoforms is determined by the amino acid sequences of their binding sites
• A change of one amino acid can be crucial to the specificity of the molecules that these enzymes metabolise

Question 19

What are the similarities shared by all CYP enzymes?

State the first similarity

Answer 19

• There are seven similarities that all CYPs share
  1. Exist in a lipid-rich environment so tend to be located in the lipophillic membrane of the smooth ER, positioned within membranes so their access channels can be easiliy accessed by lipophillic substrates

Question 20

What are the similarities shared by all CYP enzymes?

State the second similarity

Answer 20

• CYPs have highly conserved rigid regions within their active sites where iron containing haem groups reside
  
  • these are surrounded by more flexible binding regions

Question 21

What are the similarities shared by all CYP enzymes?

State the third similarity

Answer 21

• The binding area is variable and so determines the specificity of the substrate

Question 22

What are the similarities shared by all CYP enzymes?

State the fourth similarity

Answer 22

• The ability of the iron within the active site to gain or lose electons is the reason that CYPs can catalyse oxidation and reduction reactions

Question 23

What are the similarities shared by all CYP enzymes?

State the fifth similarity

Answer 23

• All CYPs require a redox partner that supplies them with electrons for their catalytic activities, i.e. P450 oxidoreductase and cytochrome b₅

Question 24

What are the similarities shared by all CYP enzymes?

State the sixth and seventh similarities

Answer 24

• The metabolic processes they catalyse require the binding and activation of oxygen
• They can all perform reduction reactions

Question 25

What are CYPs part of and why is this necessary?

How is this used to classify CYPs?

Answer 25

• CYP450s are part of a multi-component electron transfer chain, the P450-containing systems
• In order to function, CYPs require a protein partner which is responsible for the deliver y of electrons to reduce the iron in the haem and ultimately lead to molecular oxygen
• CYPs can be classified into several groups based on the type of electron transfer proteins

Question 26

State the name of the CYP systems as classified based on the type of electron transfer proteins

Answer 26

• Microsomal P450 systems
• Mitochondrial P450 systems
• Bacterial P450 systems
• CYB5 reductase/cyb5/P450 systems
• FMN/Fd/P450 systems

Question 27

Outline the microsomal P450 systems

Answer 27

• Electrons are transferred from NADPH via cytochrome P450 reductase
  
  • Cytochrome b5(cyb5) can also supply electrons to this system

Question 28

Outline the mitochondrial P450 systems

Answer 28

• Mitochondrial P450 systems includes ferredoxin reductase and ferredoxin for the transfer of electrons from NADPH to P450

Question 29

Outline the bacterial P450 systems

Answer 29

• Bacterial P450 systems employ soluble ferredoxin reductase and a ferredoxin for the transfer of electrons to CYTP450
• These proteins are located in the cytosol or closely associated with membranes but are NOT anchored within the membrane

Question 30

Outline the CYB5 reductase/cyb5/P450 systems

Answer 30

• In the CYB5 reductase/cyb5/P450 systems both electrons required by the CYP are obtained from cytochrome b5
• This system is invovled in xenobiotic metabolism and fatty acid desaturation

Question 31

Outline the FMN/Fd/P450 systems

Answer 31

• This is a one component P450 system with an FMN domain containing reductase fused to the CYP

Question 32

Outline the P450 only sytems

Answer 32

• they do not require external reducing power and are therefore not associated with any partner proteins, e.g. CYP5 (thromboxane synthase

Question 33

What are the main redox partners used by CYP450?

Answer 33

• There are two main redox partners used by CYP450 to pass electrons required for oxidation reactions

1. Cytochrome P450 monooxygenase
2. Cytochrome P450 reductase

• Cytochrome b5 has also been postulated to be inolved in the transfer of electrons to the P450 system

Question 34

Describe the redox partner cytochrome P450 monooxygenase

Answer 34

• these are found closely associated with the inner mitochondrial membrane or endoplsamic reticulum
• they can metabolise thousands of chemicals which are produced endogenously or introduced from the external environment
• Can be very specific and metabolise one substrate, e.g. CYP19(aromatase) which converts testosterone to oestradiol
• Other enzymes have many substrates, e.g. CYP3A4 which targets many drugs

Question 35

Describe the redox partner cytochrome P450 reductase

Answer 35

• These are membrane bound and are responsible for the electron transfer required for the oxidation reactions catalysed by the P40 enzymes
• They contain both FMN ad FAD binding domains that are responsible for electron flow from the NADPH-domain to the P450 enzyme

Question 36

What how may the potential redox partner cytochrome b5 be associated with CYP450?

Answer 36

• Cytochrome b5 bas been postulated to be involved in the transfer of electrons to the P450 system
• When a substrate binds to the CYP450 it results in the CYP450 becoming coupled to a redox partner protein complex P450 oxidoreductase which in turn is closely associated with cytochrome b5

Question 37

Outline the catalytic cycle of CYP450

Answer 37

• CYP450s undergo catalytic cycles which require two electrons to complete the cycle, these electrons are supplied by the oxidoreductase and cyt b5 (see figure below)

1. Substrate binds to the Fe³⁺ form of CYP450
2. Substrate is reduced the e^- transfer from NADPH-cytochrome P450 reductase
3. Molecular oxygen binds to Fe²⁺
4. Slow conversion of Fe²⁺-O₂ to the more stable Fe³⁺-O₂
5. Second reduction occurs with electron from cty b5
6. Molecular rearrangement
7. transfer of oxygen onto substrate
8. Release of product and water
   
   1. Regeneration of ferric (Fe³⁺) CYP450

Question 38

Draw a diagram of the CYP catalytic cycle

Answer 38

Question 39

Describe, in detail, phase I of xenobiotic metabolism and the reactions involved

Answer 39

• In phase I xenobiotics are acted upon by a range of enzymes that alter the substrate structure by introducing reactive and polar groups
• Reactions that occur in phase I include oxidation, reduction, hydrolysis, cyclization and decyclization
  
  • These are carried out by mixed function oxidases often in the liver
• Oxidations are most common and occurs at carbon, sulphur or nitrogen centres
• In some cases oxidation produces a sufficiently polar metabolite which may be easily excreted without further modification

Question 40

Why is phase I xenobotic metabolism often insufficient and why is phase II metabolism required?

Answer 40

• Phase I makes the xenobiotics slightly more water soluble but often this is insufficient to ensure they are retained within the urine
• Additionally pharmacological activity is not always reduced so further metabolism is required

Question 41

Describe, in detail, the phase II metabolism of xenobiotics

Answer 41

• In phase I modificiations make molecules targets for attachment of very-water soluble conugates in phase II - phase II where conjugation reactions take place
• Water-soluble conjugates include glutathione (GSH), sulfate, glycine or glucuronic acid that can bind to carboxyl (-COOH) / hydroxyl (-OH) / amino (-NH₂) / sulfhydryl (-SH) groups which were introduced into the molecule during phase I
• catalysed by large group of broad-specificity transferases, one of the most important of which is glutathione S-transferases

Question 42

What is the significance of the conjugations that occu in phase II metabolism?

Answer 42

• Products of conjugation are more hydrophilic and the increased MW makes the drug less pharmacologically or physiologically active than their substrates

Question 43

Describe, in detail, phase III of xenobiotic metabolism and why it is needed

Answer 43

• After phase II, xenobiotics must be transported out of cells into the interstitial space against a concentration gradient
• Transport is difficult because the xenobiotic now has a high molecular wieght and is hydrophobic and the membrane is lipophillic
• Efflux pumps, powered by ATP, transport the xenobiotic against its concentration gradient
  
  • There are different types of efflux transporter which form a family of ATP-binding cassette transporters

Question 44

Name two types of efflux transporter

Answer 44

• P-glycoproteins
• Multidrug resistance proteins (MRP) family

Question 45

Outline the P-glycoprotein efflux transporter and how they are important in CYP450

Answer 45

• P-glycoproteins are 170kDa transmmbrane proteins with a wide range of substrate specificity
• they are a ATP-binding cassete transporter as are all efflux transporters
• Are expressed in high levels in the gut and low level epxression in hepatocytes
• Their role is to form a barrier to xenobiotic entry by working in tandem with CYP3A
• Located on the apical side (lumen side) of cells

Question 46

Outline the multidrug resistance protein family as efflux transporters and how they are involved in CYP450

Answer 46

• The multidrug resistance protein (MRP) family and are ATP-binding cassette transporters
• MRP are responsible for transporting bulky lipophillic metabolites like GSH conjugates, steroid glucuronides and sulphated metabolites
• They are expressed in most tissues including the liver, kidney, brain
• They work in a co-ordinated manner with GST enzymes

Question 47

Give a typical substate for P-glycoproteins and how the location of the transporter is important in how it carries out its function

Answer 47

• A typical substrate for P-glycoproteins (Pgp) is digoxin which is a cardiac glycoside that is removed from the body through renal excretion
• Pgp is located on the apical (lumen) side of the membrane and ensures transepithelial transport from the blood to the lumen side of the kidney tubules

Question 48

Give an example of how a prodrug is affected by CYP450 system

Answer 48

• CYP450 monooxygenase is a important player in regulating pharmacologial activity
• In some cases a low activity pro-drug can be converted by metabolism to an active compound
  
  • Salicin is cleaved by esterases to release salicylic acid (aspirin

Question 49

How can the activiy of CYP450 enzymes be affected? Briefly list them

Answer 49

• The activity of CYP450 enzymes can be affected by
  
  • Chemical contaminants and therapeutic drugs
  • Pathologicl and physiologcal factors
  • Genetic variations called polymorphisms

Question 50

Explain, and give an example, of how chemicals contaminant and therapeutic drugs affect the activity of CYP450 enzymes

Answer 50

• They may induce or inhibit drug metabolising enzymes
• A chemical that induces an enzyme will increase its cellular expression over days through increased transcription
  
  • Polycyclic aromatic hydrocarbons in cigarette smoke can induce CYP1A
  • This has the effect of increasing drug metabolism and reducing its pharmacological activity
• Other chemicals inhibit CYP450s by direct competition at the active site, e.g. cimetidine inhibits CYP1A, instead of expression changes

Question 51

How can pathological and physiological factors affect the CYP450 system?

Answer 51

• Pathological and physiological factors affect drug metabolism
  
  • Factors include: age, enterohepatic circulation, nutrition, intestinal flora, sex differences, liver / kidney / cardiac diseases
• Age is a consideration whereby drug metabolism is found to be slower in foetal, neonate or elderly humans

Question 52

How do genetic variation affect durg activity?

Answer 52

• Genetic variations called polymorphisms afect drug activity
• A common phase II reaction involves the enzyme N-acetyltransferase
  
  • Amongst populations there are slow acetylators and fast acetylators who metabolise drugs at different rates
• This can lead to toxicity in slow acetylators or lack of response to a drug in fast acetylators

Question 53

Describe the effect rugs might have on CYP isoenzymes and how this may affect the metabolism of other drugs

How may these effects be avoided?

Answer 53

• Drugs can cause decrease in activity of some CYP isoenzymes, by competitively inhibiting CYP activity, or an increase in activity by inducing biosynthesis of the enzyme
• One drug could inhibit the metabolism of another drug resulting in accumulation and toxicity of the drug
• Makes it necessary to make dosage adjustments or select drugs that don’t interact with CYP enzymes
  
  • Important consideration when drug is vitally important to patient

Question 54

Give examples of how drugs can affect the metabolism of other drugs through CYP450

Answer 54

• Anti-epileptic drug phenyltoin induces CYP1A1, CYP2C9, CYP2C19 and CYP3A4 due to phenyltoin binding to pregnane X receptor (PXR) which results in increased transcription of CYP3A4 mRNA - this usually takes place over days
• CYP3A4 is responsible for the metabolism of over 50% of all medicines, amiodarone is a substrate and phenyltoin causes plasma levels of amiodarone to decrease and lower its pharmacological activiy

Question 55

How can induction of CYP405 enzymes have undesirable consequences?

Answer 55

• It is possible that induction results in increased toxicity if the enzyme is responsible for increased production of toxic metabolites